Huwentoxin-iv variants and methods of use

ABSTRACT

The present invention relates to Huwentoxin-IV variants, polynucleotides encoding them, and methods of making and using the foregoing.

CROSS-REFERENCE TO EARLIER APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/648,871, filed 18 May, 2012 and U.S. Provisional Application No. 61/702,538, filed 18 Sep. 2012, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to Huwentoxin-IV variants, polynucleotides encoding them, and methods of making and using the foregoing.

BACKGROUND OF THE INVENTION

Voltage-gated sodium channels (VGSC) are present in all excitable cells including cardiac and skeletal muscle cells and central and peripheral neurons. In neuronal cells, sodium channels are responsible for amplifying sub-threshold depolarizations and generating the rapid upstroke of the action potential. As such, sodium channels are essential to the initiation and propagation of electrical signals in the nervous system. Aberrant sodium channel function is thought to underlie a variety of medical disorders (Hubner and Jentsch, Hum Mol Genet. 11:2435-45, 2002) including epilepsy (Yogeeswari et al., Curr Drug Targets 5:589-602, 2004), arrhythmia (Tfelt-Hansen et al., J Cardiovasc Electrophysiol 21:107-15, 2010) myotonia (Cannon and Bean, J Clin Invest 120:80-3, 2010), and pain (Cregg et al., J Physiol 588:1897-904, 2010). Sodium channels are typically a complex of various subunits, the principle one being the pore-forming alpha-subunit, which is alone sufficient for function.

Nine known members of the family of voltage-gated sodium channel (VGSC) alpha subunits exist in humans, Nav1.1-Nav1.9. The Nav1.x subfamily can be pharmacologically subdivided into tetrodotoxin (TTX)-sensitive or TTX-resistant. Nav1.7, (also named as PN1, SCN9A or hNE) is TTX-sensitive and is primarily expressed in peripheral sympathetic and sensory neurons.

Nav1.7 function is implicated in various pain states, including acute, inflammatory and/or neuropathic pain. In man, gain of function mutations of Nav1.7 have been linked to primary erythermalgia (PE), a disease characterized by burning pain and inflammation of the extremities (Yang et al., J Med Genet. 41:171-4, 2004), and paroxysmal extreme pain disorder (PEPD) (Fertleman et al., Neuron 52:767-74, 2006). Consistent with this observation, non-selective sodium channel blockers lidocaine, mexiletine and carbamazepine can provide symptomatic relief in these painful disorders (Legroux-Crespel et al., Ann Dermatol Venereol 130:429-33, 2003; Fertleman et al., Neuron 52:767-74, 2006).

Loss-of-function mutations of SNC9A in humans cause congenital indifference to pain (CIP), a rare autosomal recessive disorder characterized by a complete indifference or insensitivity to painful stimuli (Cox et al., Nature 444:894-8, 2006; Goldberg et al, Clin Genet. 71:311-9, 2007; Ahmad et al., Hum Mol Genet. 16:2114-21, 2007).

Single nucleotide polymorphisms in the coding region of SCN9A have been associated with increased nociceptor excitability and pain sensitivity. For example, a polymorphism rs6746030 resulting in R1150W substitution in human Nav1.7 has been associated with osteoarthritis pain, lumbar discectomy pain, phantom pain, and pancreatitis pain (Reimann et al., Proc Natl Acad Sci USA 107:5148-53, 2010). DRG neurons expressing the R1150W Nav1.7 display increased firing frequency in response to depolarization (Estacion et al., Ann Neurol 66:862-6, 2009). A disabling form of fibromyalgia has been associated with SCN9A sodium channel polymorphism rs6754031, indicating that some patients with severe fibromyalgia may have a dorsal root ganglia sodium channelopathy (Vargas-Alarcon et al., BMC Musculoskelet Disord 13:23, 2012).

In mice, deletion of the SCN9A gene in nociceptive neurons lead to reduction in mechanical and thermal pain thresholds and reduction or abolition of inflammatory pain responses (Nassar et al., Proc Natl Acad Sci USA 101:12706-11, 2004). Ablating Nav1.7 gene expression in all sensory neurons abolished mechanical pain, inflammatory pain and reflex withdrawal responses to heat. Deleting SCN9A in both sensory and sympathetic neurons abolished mechanical, thermal and neuropathic pain, and recapitulated the pain-free phenotype seen in humans with SCN9A loss-of-function mutations (Minett et al., Nat Commun 3:791, 2012). Nav1.7 inhibitors or blockers may therefore be useful in the treatment of a wide range of pain associated with various disorders.

Spider venoms are known to contain a large number of sodium channel blocking peptides, including Huwentoxin-IV (HwTx-IV) (Peng et al., J Biol Chem 277:47564-71, 2002), Protoxin I, Protoxin II (Middleton et al., Biochemistry 41:14734-47, 2002) and Phrixotoxin-III (Bosmans et al., Mol Pharmacol 69:419-29, 2006). Huwentoxin-IV (HWTx-IV), from the Chinese bird spider Ornithoctonus huwena, is a potent blocker of Nav1.7 and other TTX-sensitive voltage-gated sodium channels and likely functions as a gating modifier by trapping the voltage sensor of domain II in an inward, closed conformation (Xiao et al., J Biol Chem 283:27300-13, 2008). There is a need for identification of additional Nav1.7 blockers for treatment of a wide range of pain indications. In particular, there is a need for new Nav1.7 blockers with selectivity for Nav1.7 over other VGSC isoforms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a) and b) shows IC₅₀ values for inhibition of veratridine-induced membrane depolarization for Nav1.7 for generated Huwentoxin-IV variants having specific substitutions at designated residue positions. Reference Huwentoxin-IV residue corresponds to residues in polypeptide of SEQ ID NO: 267. Substitutions highlighted in gray result in variants having IC₅₀ values ≦300×10⁻⁹ M. Values beginning with > indicate that the particular variant was inactive at the concentration indicated.

FIG. 2 a) and b) shows IC₅₀ values for inhibition of veratridine-induced membrane depolarization for Nav1.2 for generated Huwentoxin-IV variants having specific substitutions at designated residue positions. Reference Huwentoxin-IV residue corresponds to residues in polypeptide of SEQ ID NO: 267. Values beginning with > indicate that the particular variant was inactive at the concentration indicated.

FIG. 3 a) and b) shows selectivity of generated Huwentoxin-IV variants as ratios of IC₅₀ values for Nav1.2 to IC₅₀ values for Nav1.7 for each variant having specific substitutions at designated residue positions (IC₅₀ values calculated for inhibition of veratridine-induced membrane depolarization). Reference Huwentoxin-IV residue corresponds to residues in polypeptide of SEQ ID NO: 267. Substitutions highlighted in gray result in variants having IC₅₀ (Nav1.2)/IC₅₀ (Nav1.7) ratio equal or over 5.0. Values beginning with > indicate that the particular variant was inactive at the concentration indicated. “Inact” indicates that the peptide was inactive on Nav1.7.

FIG. 4 shows sequences of Huwentoxin-IV variants having EC₅₀ for Nav1.7≦300×10⁻⁹M (IC₅₀ values calculated for inhibition of veratridine-induced membrane depolarization).

FIG. 5 shows sequences of Huwentoxin-IV variants that are at least 5-fold more selective for Nav1.7 than Nav1.2, assessed using the IC₅₀ (Nav1.2)/IC₅₀ (Nav1.7) ratio, of are inactive at Nav1.2 (IC₅₀ values calculated for inhibition of veratridine-induced membrane depolarization).

FIG. 6 shows IC₅₀ values and selectivity for select Huwentoxin-IV variants in whole cell patch-clamp assay (QPatch).

FIG. 7 shows line graph of Randall-Selitto paw pressure thresholds in grams (g) before (Pre) and 5, 10, 20, 30, 45 and 60 minutes following dorsal hind paw injections of vehicle (n=9) or a) 0.3 nmoles, b) 3 nmoles or c) 30 nmoles of huwentoxin IV (n=9) in rat. Data are represented as mean±s.e.m using Two-way ANOVA with Bonferroni post-tests. NS=not significant; **=p<0.01; ***=p<0.001.

FIG. 8 shows mean area under the curve (AUC) of gram thresholds for huwentoxin IV-treated rats with subtraction (for each individual huwentoxin IV-treated rat) of the mean AUC for vehicle-treated animals. Using one-way ANOVA, there was a significant effect of dose (p<0.001) demonstrating dose-dependent responses. Bonferroni post tests showed significant differences between each dose group, **=p<0.01; ***=p<0.001.

FIG. 9 shows various Huwentoxin-IV alanine mutants that cause significant (>10×) reduction in function (QPatch) colored by average per residue C-alpha (CA) atoms root mean square deviation (RMSD) calculated from their respective molecular dynamics simulations (50 ns each). The CA RMSDs are colored on a gradient from 0.5 Å in red to 2.2 Å in blue. (a) WT (b) F6A, (c) P11A, (d) D14A, (e) L22A, (f) S25A, (g) W30A, (h) K32A and (i) Y33A Huwentoxin-IV mutants.

FIG. 10 shows various Huwentoxin-IV alanine mutants that appear to cause isoform specific changes in function (QPatch) colored by average per residue CA RMSD calculated from their respective molecular dynamics simulations (50 ns each). The CA RMSDs are colored on a gradient from 0.5 Å in red to 2.2 Å in blue. (a) K18A, (b) R26A, (c) K27A.

FIG. 11 shows NMR solution structure of recombinant Huwentoxin-IV (SEQ ID NO:1). The NMR structure reveals 5 residues in HwTx-IV (F6, T28, W30, K32 and Y33) that form a twisted β-sheet (cyan) to create a polar-aryl face, a putative interacting surface between Huwentoxin-IV and Nav1.7.

FIG. 12 shows Homology model of the domain 2 (DII) voltage sensing domain (VSD) of hNav1.7 with Huwentoxin-IV docked. Based on this model, Huwentoxin-IV docks in a grove made by segments S1-S2 and S3-S4. Huwentoxin-IV (SEQ ID NO:1) residues K32 and W30 are predicted to interact with Nav1.7 (SEQ ID NO:263) residues E811 and M750, respectively.

FIG. 13 shows A) sequences and B) IC₅₀ values for NV1D2168 (SEQ ID NO: 102) variants.

FIG. 14 shows A) sequences and B) IC₅₀ values for NV1D2163 (SEQ ID NO: 3) variants.

SUMMARY OF THE INVENTION

One embodiment of the invention is an isolated Huwentoxin-IV variant comprising a sequence X₁CX₂X₃X₄FX₅X₆CX₇X₈X₉X₁₀X₁₁X₁₂CCX₁₃X₁₄X₁₅X₁₆X₁₇X₁₈CX₁₉X₂₀X₂₁X₂₂X₂₃X₂₄CKX₂₅X₂₆IX₂₇X₂₈ (SEQ ID NO: 265); wherein X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄, X₁₅, X₁₆, X₁₇, X₁₈, X₁₉, X₂₀, X₂₁, X₂₂, X₂₃, X₂₄, X₂₅, and X₂₆ are any amino acid; X₂₇ and X₂₈ are any amino acid or deleted; and the Huwentoxin-IV variant has an IC₅₀ value about 300×10⁻⁹ M or less for human Nav1.7 (SEQ ID NO: 263), with the proviso that the Huwentoxin-IV variant is not a polypeptide comprising a sequence shown in SEQ ID NO: 1.

Another embodiment of the invention is an isolated Huwentoxin-IV variant comprising a sequence X₁CX₂X₃X₄FX₅X₆CX₇X₈X₉X₁₀X₁₁X₁₂CCX₁₃X₁₄X₁₅X₁₆X₁₇X₁₈CX₁₉X₂₀X₂₁X₂₂X₂₃X₂₄CKX₂₅X₂₆IX₂₇X₂₈ (SEQ ID NO: 265); wherein X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄, X₁₅, X₁₆, X₁₇, X₁₈, X₁₉, X₂₀, X₂₁, X₂₂, X₂₃, X₂₄, X₂₅, and X₂₆ are any amino acid; X₂₇ and X₂₈ are any amino acid or deleted; and the Huwentoxin-IV variant selectively inhibits Nav1.7, with the proviso that the Huwentoxin-IV variant is not a polypeptide comprising a sequence shown in SEQ ID NO: 1.

Another embodiment of the invention is an isolated Huwentoxin-IV variant comprising a sequence X₁CX₂X₃X₄FX₅X₆CX₇X₈X₉X₁₀X₁₁X₁₂CCX₁₃X₁₄X₁₅X₁₆X₁₇X₁₈CX₁₉X₂₀X₂₁TX₂₂ WCKYX₂₃X₂₄X₂₅X₂₆ (SEQ ID NO: 276); wherein X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄, X₁₅, X₁₆, X₁₇, X₁₈, X₁₉, X₂₀, X₂₁, X₂₂, X₂₃ and X₂₄ are any amino acid; X₂₅ and X₂₆ are any amino acid or deleted; and the Huwentoxin-IV variant has an IC₅₀ value about 300×10⁻⁹ M or less for human Nav1.7 (SEQ ID NO: 263), with the proviso that the Huwentoxin-IV variant is not a polypeptide comprising a sequence shown in SEQ ID NO: 1.

Another embodiment of the invention is an isolated polynucleotide encoding the Huwentoxin-IV variants of the invention.

Another embodiment of the invention is a vector comprising the isolated polynucleotides of the invention.

Another embodiment of the invention is a host cell comprising a vector of the invention.

Another embodiment of the invention is a method of producing the isolated Huwentoxin-IV variant polypeptide of the invention comprising culturing the host cell of the invention and recovering the Huwentoxin-IV variant polypeptide by the host cell.

Another embodiment of the invention is a pharmaceutical composition comprising the isolated Huwentoxin-IV variant of the invention and a pharmaceutically acceptable excipient.

Another embodiment of the invention is a method of treating pain in a subject, comprising administering to the subject an effective amount of the Huwentoxin-IV variant of the invention to treat pain, other disorders of sensory or sympathetic neuron dysfunction.

DETAILED DESCRIPTION OF THE INVENTION

All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as though fully set forth.

As used herein and in the claims, the singular forms “a,” “and,” and “the” include plural reference unless the context clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which an invention belongs. Although any compositions and methods similar or equivalent to those described herein can be used in the practice or testing of the invention, exemplary compositions and methods are described herein.

The term “polypeptide” means a molecule that comprises at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides of less than 50 amino acids may be referred to as “peptides”. Polypeptides may also be referred as “proteins”.

The term “polynucleotide” means a molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. Double and single-stranded DNAs and RNAs are typical examples of polynucleotides.

The term “complementary sequence” means a second isolated polynucleotide sequence that is antiparallel to a first isolated polynucleotide sequence and that comprises nucleotides complementary to the nucleotides in the first polynucleotide sequence.

The term “vector” means a polynucleotide capable of being duplicated within a biological system or that can be moved between such systems. Vector polynucleotides typically contain elements, such as origins of replication, polyadenylation signal or selection markers, that function to facilitate the duplication or maintenance of these polynucleotides in a biological system. Examples of such biological systems may include a cell, virus, animal, plant, and reconstituted biological systems utilizing biological components capable of duplicating a vector. The polynucleotides comprising a vector may be DNA or RNA molecules or hybrids of these.

The term “expression vector” means a vector that can be utilized in a biological system or a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

The term “wild type Huwentoxin-IV” or “wild type HwTx-IV” as used herein refers to Chinese bird spider Ornithoctonus huwena Huwentoxin-IV polypeptide having a sequence shown in SEQ ID NO: 1 (ECLEIFKACNPSNDQCCKSSKLVCSRKTRWCKYQI). The term “recombinant Huwentoxin-IV” or recombinant HwTx-IV” as used herein refers to the recombinantly expressed Huwentoxin-IV having a sequence shown in SEQ ID NO: 2 (GPECLEIFKACNPSNDQCCKSSKLVCSRKTRWCKYQIGK). Recombinant Huwentoxin-IV incorporates a two amino acid N- and C-terminal tail when compared to the wild type Huwentoxin-IV. The term “reference Huwentoxin-IV” refers to a polypeptide sequence of SEQ ID NO: 267 (ECLEIFKACNPSNDQCCKSSKLVCSRKTRWCKYQIGK). Throughout the specification, residue numbering is according to SEQ ID NO: 267. For example, “F6” in the specification refers to Phenylalanine residues at position 6 of SEQ ID NO: 267.

The term “variant” as used herein refers to a polypeptide or a polynucleotide that differs from the wild type Huwentoxin-IV polypeptide of SEQ ID NO: 1 or the wild type Huwentoxin-IV polynucleotide of SEQ ID NO: 268 sequence by one or more modifications for example, substitutions, insertions or deletions of nucleotides or amino acids.

“Nav1.7” (also called as SCN9A, hNE, PN1) as used herein refers to the well known sodium channel protein type 9 subunit alpha having a sequence shown in GenBank accession number NP_(—)002968.1 and in SEQ ID NO: 263.

“Nav1.2” as used herein refers to the well known sodium channel protein type 2 subunit alpha (SCN2A) having a sequence shown in GenBank accession number NP_(—)001035232.1 and in SEQ ID NO: 264.

“Blocks activity” or “inhibits activity” as used herein refers to an ability of Huwentoxin-IV variants to reduce membrane depolarization induced by veratridine (3-Veratroylveracevine) by at least 100, 200, 300, 400, 500, 60%, 70%, 75%, 80%, 85%, 90%, 95% or 100% in an in vitro membrane depolarization assay using FRET (fluorescence resonance energy transfer), where veratridine-induced depolarization is measured as a reduction in FRET signal using DISBAC2(3) ([bis-(1,3-diethylthiobarbituric acid)trimethine oxonol]) as an acceptor and PTS18 (trisodium 8-octadecyloxypyrene-1,3,6-trisulfonate) as a donor by exciting the donor at 390-420 nm and measuring FRET at 515-575 nm using cell lines stably expressing Nav1.7.

The present invention provides isolated Huwentoxin-IV (HwTx-IV) variant polypeptides that inhibit Nav1.7, polynucleotides encoding them, vectors, host cells, and methods of using the polynucleotides and polypeptides of the invention. The variants of the invention may be more potent or more selective towards Nav1.7 when compared to the recombinant Huwentoxin-IV polypeptide. The polypeptides of the invention inhibit depolarization resulting from Nav1.7 activation, and therefore may be useful in the treatment of various conditions associated with pain and conditions associated with sensory or sympathetic neuron dysfunction. The current invention is based, at least in part, on the finding that certain residues in Huwentoxin-IV are intolerant to substitutions, specifically F6, K32 and I35, and additionally residues I5, P11, D14, S25, K27, T28, W30 and Y33 (residue numbering according to SEQ ID NO: 267) are substantially intolerant to substitutions, while other residues may be substituted to enhance potency and/or selectivity of Huwentoxin-IV variants for Nav1.7 as long as the cysteine residues at positions C2, C9, C16, C17, C24 and C31 remain intact.

One embodiment of the invention is an isolated Huwentoxin-IV variant comprising a sequence

-   -   X₁CX₂X₃X₄FX₅X₆CX₇X₈X₉X₁₀X₁₁X₁₂CCX₁₃X₁₄X₁₅X₁₆X₁₇X₁₈CX₁₉X₂₀X₂₁X₂₂X₂₃X₂₄CKX₂₅X₂₆IX₂₇X₂₈         (SEQ ID NO: 265); wherein     -   a) X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀X₁₁, X₁₂, X₁₃X₁₄, X₁₅,         X₁₆, X₁₇X₁₈, X₁₉, X₂₀X₂₁, X₂₂X₂₃, X₂₄, X₂₅, and X₂₆ are any         amino acid;     -   b) X₂₇ and X₂₈ are any amino acid or deleted; and     -   c) the Huwentoxin-IV variant has an IC_(H) value about 300×10⁻⁹         M or less for human Nav1.7 (SEQ ID NO: 263), with the proviso         that the Huwentoxin-IV variant is not a polypeptide comprising a         sequence shown in SEQ ID NOs: 1.

The Huwentoxin-IV variants of the invention are equally potent or more potent Nav1.7 inhibitors when compared to recombinant Huwentoxin-IV (SEQ ID NO: 2). Recombinant Huwentoxin-IV has an IC₅₀ value of about 160×10⁻⁹ M for human Nav1.7 in a veratridine-induced depolarization inhibition assay measuring decline in FRET (fluorescence resonance energy transfer) in cells stably expressing Nav1.7 using FLIPR® Tetra instrument (Molecular Devices). A Huwentoxin-IV variant is “equally potent or more potent” Nav1.7 inhibitor when the IC₅₀ value in the assay described above is about 300×10⁻⁹ M or less. This IC₅₀ value is set higher than the measured IC₅₀ for the recombinantly expressed Huwentoxin-IV due to the intrinsic variability (½ log) of the assay itself. For clarity, an IC₅₀ of 300×10⁻⁹ M is identical to IC₅₀ of 3.0×10⁻⁷ M.

The Huwentoxin-IV variants of the invention retain the native disulfide bridges between C2-C17, C9-C24 and C16-C31 in addition to invariant residues F6, K32 and I35 (residue numbering according to SEQ ID NO: 267), while the remaining residues can be substituted with any amino acid as long as the resulting variant in the above Nav1.7 inhibition assay has an IC₅₀ of about 300×10⁻⁹ M or less.

The Huwentoxin-IV variant polypeptides of the invention may be produced by chemical synthesis, such as solid phase peptide synthesis, on an automated peptide synthesizer. Alternatively, the polypeptides of the invention can be obtained from polynucleotides encoding the polypeptides by the use of cell-free expression systems such as reticulocyte lysate based expression systems, or by standard recombinant expression systems. Those skilled in the art will recognize other techniques for obtaining the polypeptides of the invention. In an exemplary method, the Huwentoxin-IV variants of the invention are generated by expressing them as human serum albumin (HSA) fusion proteins utilizing a glycine-rich linker such as (GGGGS)₄ (SEQ ID NO: 269) or (GGGGS)₆ (SEQ ID NO: 266) coupled to a protease cleavable linker such as a recognition sequence for HRV3C protease (Recombinant type 14 3C protease from human rhinovirus) LEVLFQGP (HRV3C linker) (SEQ ID NO: 270)). Hexahistidine or other tags may be used to facilitate purification using well known methods.

Generation of the Huwentoxin-IV variants is typically achieved at the nucleic acid level. The polynucleotides can be synthesized using chemical gene synthesis according to methods described in U.S. Pat. No. 6,521,427 and U.S. Pat. No. 6,670,127, utilizing degenerate oligonucleotides to generate the desired variants, or by standard PCR cloning and mutagenesis. Libraries of variants can be generated by standard cloning techniques to clone the polynucleotides encoding the Huwentoxin-IV variants into the vector for expression.

The Huwentoxin-IV variants may incorporate additional N- and/or C-terminal amino acids when compared to the wild type HwTx-IV of SEQ ID NO: 1, for example resulting from cloning and/or expression schemes. For example, cleavage from HSA after expression of the variant as HSA-(GGGGS)₄-HRV3C linker-HwTx-IV variant fusion protein may result in the incorporation of additional two residues to the N-terminus of each HwTx-IV variant, such as G and P. Additional residues may be incorporated to the C-terminus of the HwTx-IV variants, such as G and K to generate an endogenous amidation recognition sequence.

The HwTx-IV variants of the invention are tested for their ability to inhibit Nav1.7 using methods described herein. An exemplary assay is a veratridine-induced depolarization inhibition assay measuring decline in FRET (fluorescence resonance energy transfer) in cells stably expressing Nav1.7. Another exemplary assay employs electrophysiological recordings to measure the total influx of sodium ions (Na⁺) across the cell membrane by way of voltage differential using well known patch clamp techniques and described herein.

In another embodiment, an isolated Huwentoxin-IV variant comprises a sequence

-   -   X₁CX₂X₃X₄FX₅X₆CX₉X₈X₉X₁₀X₁₁X₁₂CCX₁₃X₁₄X₁₅X₁₆X₁₉X₁₈CX₁₉X₂₀X₂₁X₂₂X₂₃X₂₄CKX₂₅X₂₆IX₂₇X₂₉         (SEQ ID NO: 265); wherein     -   a) X₄ is Y, V or I;     -   b) X₈ is P or V;     -   c) X₁₁ is D, P or W;     -   d) X₁₉ is S or I;     -   e) X₂₁ is Y, W, A, H or K;     -   f) X₂₂ is T or V;     -   g) X₂₄ is W or K;     -   h) X₂₅ is W, T, I or Y;     -   i) X₁, X₂, X₃, X₅, X₆, X₇, X₉, X₁₀, X₁₂, X₁₃, X₁₄, X₁₅, X₁₆,         X₁₇, X₁₈, X₂₀, X₂₃ and X₂₆ are any amino acid;     -   j) X₂₇ and X₂₈ are any amino acid or deleted; and     -   k) the Huwentoxin-IV variant has an IC₅₀ value about 300×10⁻⁹ M         or less for human Nav1.7 (SEQ ID NO: 263), with the proviso that         the Huwentoxin-IV variant is not a polypeptide comprising a         sequence shown in SEQ ID NO: 1.

In another embodiment, the isolated Huwentoxin-IV variant comprises a sequence

-   -   X₁CX₂X₃X₄FX₅X₆CX₇X₈X₉X₁₀X₁₁X₁₂CCX₁₃X₁₄X₁₅X₁₆X₁₇X₁₈CX₁₉X₂₀X₂₁X₂₂X₂₃X₂₄CKX₂₅X₂₆IX₂₉X₂₉         (SEQ ID NO: 265); wherein     -   a) X₁ is K, R, H, D, Y, F, N, Q, S, T, G, L, I, P or E;     -   b) X₂ is R, F, W, N, S or L;     -   c) X₃ is R, H, D, Y, N, Q, L, I, P or E;     -   d) X₄ is Y, V or I;     -   e) X₅ is R, W, Q, S or K;     -   f) X₆ is R, E, Y, F, V or A;     -   g) X₇ is K, R, E, Y, F, S, V or N;     -   h) X₈ is P or V;     -   i) X₉ is R, F, Q, V or S;     -   j) X₁₀ is H, D, Y, W, Q, S, T, G, A, V, L, I, P or N;     -   k) X₁₁ is D, P or W;     -   l) X₁₂ is K, R, D, E, Y, W, N, T, A, L or Q;     -   m) X₁₃ is R, Y, Q, S, T, G, L, I, P or K;     -   n) X₁₄ is K, R, Y, F, N, Q, G, A, V, L, I, P or S;     -   o) X₁₅ is R, H, D, Y, W, N, Q, T, V, I, P or S;     -   p) X₁₆ is R, H, D, F, W, N, Q, S, T, G, A, L or K;     -   q) X₁₇ is K, R, Y, F, W, P or L;     -   r) X₁₈ is K, R, T, A, L or V;     -   s) X₁₉ is S or I;     -   t) X₂₀ is K, W, G, A, I, D or R;     -   u) X₂₁ is Y, W, A or K;     -   v) X₂₂ is T or V;     -   w) X₂₃ is K, H, W, N, G, A, L or R;     -   x) X₂₄ is W or K;     -   y) X₂₅ is W, T, I or Y;     -   z) X₂₆ is K, R, Y, F, S, T, G, A, V, L, I or Q;     -   aa) X₂₇ is K, R, H, F, W, V, L, I, G or deleted; and     -   bb) X₂₈ is R, H, Y, F, W, N, G, V, P, K or deleted; and the         Huwentoxin-IV variant has an IC₅₀ value about 300×10⁻⁹ M or less         for human Nav1.7 (SEQ ID NO: 263), with the proviso that the         Huwentoxin-IV variant is not a polypeptide comprising a sequence         shown in SEQ ID NO: 1.

The Huwentoxin-IV variants of the invention may inhibit Nav1.7 with an IC₅₀ value of between about 12×10⁻⁹ M to about 300×10⁻⁹ M. Exemplary variants demonstrating the range of IC₅₀ values are polypeptides of SEQ ID NOs: 3-222 shown in FIG. 4.

Another embodiment of the invention is an isolated Huwentoxin-IV variant comprising a sequence X₁CX₂X₃X₄FX₅X₆CX₇X₈X₉X₁₀X₁₁X₁₂CCX₁₃X₁₄X₁₅X₁₆X₁₇X₁₈CX₁₉X₂₀X₂₁X₂₂X₂₃X₂₄CKX₂₅X₂₆IX₂₉X₂₈ (SEQ ID NO: 265); wherein

-   -   a) X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀, X₁₁, X₁₂, X₁₃X₁₄,         X₁₅, X₁₆, X₁₇, X₁₈, X₁₉, X₂₀, X₂₁, X₂₂, X₂₃, X₂₄, X₂₅, and X₂₆         are any amino acid;     -   b) X₂₇ and X₂₈ are any amino acid or deleted; and     -   c) the Huwentoxin-IV variant selectively inhibits Nav1.7, with         the proviso that the Huwentoxin-IV variant is not a polypeptide         comprising a sequence shown in SEQ ID NO: 1.

The Huwentoxin-IV variants of the invention may be more selective towards Nav1.7 when compared to the recombinant Huwentoxin-IV (SEQ ID NO: 2). Recombinant Huwentoxin-IV has an IC₅₀ of about 159×10⁻⁹ M for Nav1.7 and an IC₅₀ of about 342×10⁻⁹ M for Nav1.2, and therefore the ratio of IC₅₀ for Nav1.2 to IC₅₀ for Nav1.7 about 2.143. “Selectivity” or “selective” or “more selective” or “selectively blocks” or “selectively inhibits” when used herein refers to a Huwentoxin-IV variant that has a ratio of IC₅₀ for Nav1.2 to IC₅₀ for Nav1.7 (IC₅₀ (Nav1.2)/IC₅₀ (Nav1.7)) equal or over about 5.0. In addition, a Huwentoxin-IV variant “selectively inhibits” Nav1.7 in instances when the variant does not inhibit Nav1.2 at a peptide concentration of at least 0.8×10⁻⁶ M even if the IC₅₀ ratio is less than 5. IC₅₀ for Nav1.2 can be assayed in a veratridine-induced depolarization inhibition assay using cell lines stably expressing Nav1.2 according to methods described for Nav1.7.

Residue positions in Huwentoxin-IV that can be mutagenized to improve selectivity include residues N13, D14, Q15, K18, S19, S20, K21, L22, R26, K27, R29, W30, Y33 and Q34 (residue numbering according to SEQ ID NO: 267). Exemplary substitutions to improve selectivity are N13G, N131, Q15E, Q15W, Q15P, K18F, K18P, S19Q, R26K and R26I. Exemplary Huwentoxin-IV variants with improved selectivity are variants of SEQ ID NOs: 5, 7, 12, 13, 16, 21, 25, 45, 46, 48, 55, 57, 58, 60, 61, 72, 74, 76, 78, 82, 83, 96, 109, 111, 113, 122, 127, 131, 134, 137, 141, 142, 149, 164, 165, 172, 175, 177, 178, 180, 182, 188, 189, 192, 198, 202, 204, 213, 215, 219, and 223-240.

Residues K7, N13, D14, Q15, K18, S19, S20, K21, L22, V23, R26, K27, R29, W30, Y33, and Q34, G36 and K37 (residue number according to SEQ ID NO: 267) may be substituted to improve both potency and selectivity of the resulting Huwentoxin-IV variants (FIGS. 1 and 3). Exemplary substitutions increasing both potency and selectivity are R26K, Y33W, G36I, N13Q, S19Q, and K37R (residue numbering according to SEQ ID NO: 267). Exemplary variants with improved potency and selectivity are variants of SEQ ID NOs: 5, 6, 7, 12, 13, 16, 21, 25, 45, 46, 48, 55, 57, 58, 60, 61, 72, 74, 76, 78, 82, 83, 96, 109, 111, 113, 122, 127, 131, 134, 137, 141, 142, 149, 164, 165, 169, 172, 175, 177, 178, 180, 181, 182, 187, 188, 189, 192, 198, 202, 203, 204, 207, 213, 215, 216, 219 and 221.

Another embodiment of the invention is a Huwentoxin-IV variant having teh amino acid sequence shown in SEQ ID NOs: 277-355.

Selectivity and/or potency of the Huwentoxin-IV variants of the invention can further be improved by selective substitutions (grafting) at positions identified to modulate selectivity and/or potency into existing variants. Exemplary variants that can further be modified and/or improved are variants NV1G387 (E1N, R26K, Q34S, G36I; NV1D2168, SEQ ID NO: 192) and NV1G327 (E1N, E4R, Y33W, Q34S; NV1D2163, SEQ ID NO: 3). NV1G387 demonstrated high selectivity towards Nav1.7. The potency of NV1G387 can be potentially improved by diversifying positions E4, A8, N13, Q15, K18, S19, S20, K21, L22, S25, K37 and G36. Exemplary substitutions are shown in FIG. 13A and FIG. 14A. NV1G327 demonstrated higher potency towards Nav1.7. The selectivity of NG1G327 can be potentially improved by diversifying positions F6, P11, D14, Q15, K18, S19, R26, K27, R29, K32 and Y33. Exemplary substitutions are shown in FIG. 13A and FIG. 14A. Those skilled in the art will recognize that substitutions at any Huwentoxin-IV variant described herein may be combined and the effect of the combination on the potency, selectivity or other characteristics can be assessed using methods described herein.

Another embodiment of the invention is an isolated Huwentoxin-IV variant comprising a sequence

-   -   X₁CX₂X₃X₄FX₅X₆CX₇X₈X₉X₁₀X₁₁X₁₂CCX₁₃X₁₄X₁₅X₁₆X₁₇X₁₈CX₁₉X₂₀X₂₁TX₂₂WCKYX₂₃X₂₄X₂₅X₂₆         (SEQ ID NO: 276); wherein         X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄,         X₁₅, X₁₆, X₁₇, X₁₈, X₁₉, X₂₀, X₂₁, X₂₂, X₂₃ and X₂₄ are any         amino acid;         X₂₅ and X₂₆ are any amino acid or deleted; and         the Huwentoxin-IV variant has an IC₅₀ value about 300×10⁻⁹ M or         less for human Nav1.7 (SEQ ID NO: 263), with the proviso that         the Huwentoxin-IV variant is not a polypeptide comprising a         sequence shown in SEQ ID NO: 1.     -   Huwentoxin-IV variant of SEQ ID NO: 276 may comprise following         substitutions:     -   X₄ is Y, V or I;     -   X₈ is P or V;     -   X₁₁ is D, P or W;     -   X₁₉ is S or I;     -   X₂₁ is Y, W, A, H or K; and     -   X₂₄ is I in SEQ ID NO: 276.     -   Huwentoxin-IV variant of SEQ ID NO: 276 may further comprise         following substitutions:     -   X₁ is K, R, H, D, Y, F, N, Q, S, T, G, L, I, P or E;     -   X₂ is R, F, W, N, S or L;     -   X₃ is R, H, D, Y, N, Q, L, I, P or E;     -   X₅ is R, W, Q, S or K;     -   X₆ is R, E, Y, F, V or A;     -   X₇ is K, R, E, Y, F, S, V or N;     -   X₉ is R, F, Q, V or S;     -   X₁₀ is H, D, Y, W, Q, S, T, G, A, V, L, I, P or N;     -   X₁₂ is K, R, D, E, Y, W, N, T, A, L or Q;     -   X₁₃ is R, Y, Q, S, T, G, L, I, P or K;     -   X₁₄ is K, R, Y, F, N, Q, G, A, V, L, I, P or S;     -   X₁₅ is R, H, D, Y, W, N, Q, T, V, I, P or S;     -   X₁₆ is R, H, D, F, W, N, Q, S, T, G, A, L or K;     -   X₁₇ is K, R, Y, F, W, P or L;     -   X₁₈ is K, R, T, A, L or V;     -   X₂₀ is K, W, G, A, I, D or R;     -   X₂₂ is K, H, W, N, G, A, L or R;     -   X₂₃ is K, R, Y, F, S, T, G, A, V, L, I or Q;     -   X₂₅ is K, R, H, F, W, V, L, I, G or deleted; and     -   X₂₆ is R, H, Y, F, W, N, G, V, P, K or deleted.

The isolated Huwentoxin-IV variant of SEQ ID NO: 276 may have an IC₅₀ of less than about 160×10⁻⁹ M for human Nav1.7.

The Huwentoxin-IV variant of SEQ ID NO: 276 may bind human Nav1.7 at residues F6, T28, W30, K32 and Y33. As long as these residues are kept invariant, other residues in the Huwentoxin-IV may be altered to improve properties, such as affinity and/or selectivity using methods described herein.

Another embodiment of the invention is an isolated fusion protein comprising the Huwentoxin-IV variant of SEQ ID NOs: 3-253 or 277-355 fused with a second polypeptide. Such second polypeptides may be leader or secretory signal sequences, partially or completely synthetic sequences resulting for example from cloning steps, or tags such as hexahistidine tag.

Additional moieties may be incorporated into the Huwentoxin-IV variants of the invention such as polyethylene glycol (PEG) molecules, such as PEG5000 or PEG20000, fatty acids and fatty acid esters of different chain lengths, for example laurate, myristate, stearate, arachidate, behenate, oleate, arachidonate, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like, polylysine, octane, carbohydrates (dextran, cellulose, oligo- or polysaccharides) for desired properties. These moieties may be direct fusions with the Huwentoxin-IV variant polypeptides and may be generated by standard cloning and expression techniques. Alternatively, well known chemical coupling methods may be used to attach the moieties to recombinantly produced HwTx-IV variants of the invention.

Huwentoxin-IV variants incorporating additional moieties may be compared for functionality by several well known assays. For example, pharmacokinetic properties of Huwentoxin-IV variants coupled to PEG may be evaluated in well known in vivo models.

Another embodiment of the invention is an isolated Huwentoxin-IV variant comprising a polypeptide sequence of SEQ ID NOs: 3-253 or 277-355.

Another embodiment of the invention is an isolated polynucleotide comprising a polynucleotide encoding the Huwentoxin-IV variant polypeptide of the invention.

The polynucleotides of the invention may be produced by chemical synthesis such as solid phase polynucleotide synthesis on an automated polynucleotide synthesizer. Alternatively, the polynucleotides of the invention may be produced by other techniques such as PCR based duplication, vector based duplication, or restriction enzyme based DNA manipulation techniques. Techniques for producing or obtaining polynucleotides of a given known sequence are well known in the art.

The polynucleotides of the invention may also comprise at least one non-coding sequence, such as transcribed but not translated sequences, termination signals, ribosome binding sites, mRNA stabilizing sequences, introns and polyadenylation signals. The polynucleotide sequences may also comprise additional sequences encoding additional amino acids. These additional polynucleotide sequences may, for example, encode a marker or well known tag sequences such as a hexa-histidine or a HA tag which facilitate the purification of fused polypeptides. Certain exemplary polynucleotides are disclosed herein, however, other polynucleotides which, given the degeneracy of the genetic code or codon preferences in a given expression system, encode the antibody antagonists of the invention are also within the scope of the invention. Exemplary polynucleotides are polynucleotides comprising a sequence shown in SEQ ID NOs: 271-275.

Another embodiment of the invention is a vector comprising an isolated polynucleotide encoding the Huwentoxin-IV variants of the invention. The vectors of the invention are useful for maintaining polynucleotides, duplicating polynucleotides, or driving expression of a polypeptide encoded by a vector of the invention in biological systems, including reconstituted biological systems. Vectors may be chromosomal-, episomal- and virus-derived such as vectors derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, baculoviruses, papova viruses such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses, picornaviruses and retroviruses and vectors derived from combinations thereof, such as cosmids and phagemids.

In one embodiment of the invention the vector is an expression vector. Expression vectors typically comprise nucleic acid sequence elements that can control, regulate, cause or permit expression of a polypeptide encoded by such a vector. Such elements may comprise transcriptional enhancer binding sites, RNA polymerase initiation sites, ribosome binding sites, and other sites that facilitate the expression of encoded polypeptides in a given expression system. Such expression systems may be cell-based, or cell-free systems well known in the art. Nucleic acid sequence elements and parent vector sequences suitable for use in the expression of encoded polypeptides are also well known. An exemplary plasmid-derived expression vector useful for expression of the polypeptides of the invention comprises an E. coli origin of replication, an ampicillin resistance (Amp) gene, a CMV promoter, a signal sequence, and a SV40 polyadenlyation site.

Another embodiment of the invention is an isolated host cell comprising a vector of the invention. Exemplary host cells include Archaea cells; bacterial cells such as Streptococci, Staphylococci, Enterococci, E. coli, Streptomyces, cyanobacteria, B. subtilis and S. aureus; fungal cells such as Kluveromyces, Saccharomyces, Basidomycete, Candida albicans or Aspergillus; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK293, CV-1, Bowes melanoma and myeloma; and plant cells, such as gymnosperm or angiosperm cells. The host cells in the methods of the invention may be provided as individual cells, or populations of cells. Populations of cells may comprise an isolated or cultured population of cells or cells present in a matrix such as a tissue.

Introduction of a polynucleotide, such as a vector, into a host cell can be effected by methods well known to those skilled in the art. These methods include calcium phosphate transfection, DEAE-Dextran mediated transfection, microinjection, cationic lipid-mediated transfection and electroporation.

Another embodiment of the invention is a method for expressing the Huwentoxin-IV variant of the invention comprising the steps of providing a host cell of the invention; and culturing the host cell under conditions sufficient for the expression of at least one Huwentoxin-IV variant of the invention.

Host cells can be cultured under any conditions suitable for maintaining or propagating a given type of host cell and sufficient for expressing a polypeptide. Culture conditions, media, and related methods sufficient for the expression of polypeptides are well known in the art. For example, many mammalian cell types can be aerobically cultured at 37° C. using appropriately buffered DMEM media while bacterial, yeast and other cell types may be cultured at 37° C. under appropriate atmospheric conditions in LB media.

In the methods of the invention the expression of the Huwentoxin-IV variant can be confirmed using a variety of well known methods. For example, expression of a polypeptide can be confirmed using detection reagents, such as antibodies using for example FACS or immunofluorescent techniques, or using SDS-PAGE or HPLC.

Another aspect of the invention is a method of modulating the activity of Nav1.7 in a biological tissue, the method comprising contacting a biological tissue expressing Nav1.7 with a Nav1.7 modulating amount of a Huwentoxin-IV variant of the invention, or a pharmaceutically acceptable salt thereof.

Methods of Treatment

Huwentoxin-IV variants of the invention may be utilized in any therapy where it is desired to treat, reduce or alleviate symptoms of pain or other disorders of sensory or sympathetic neuron dysfunction.

The methods of the invention may be used to treat an animal patient belonging to any classification. Examples of such animals include mammals such as humans, rodents, dogs, cats and farm animals.

Pain treated with the Huwentoxin-IV variants of the invention may be any type of pain, such as chronic pain, acute pain, neuropathic pain, nociceptive pain, visceral pain, back pain, pain associated with inflammatory conditions, post-operative pain, thermal pain or pain associated with disease and degeneration.

The pain may result from one or more causes, such as peripheral neuropathy, central neuropathy, nerve compression or entrapment syndromes such as carpal tunnel syndrome, tarsus tunnel syndrome, ulnar nerve entrapment, compression radiculopathy, lumbar spinal stenosis, sciatic nerve compression, spinal root compression, intercostal neuralgia, compression radiculopathy and radicular lower back pain, spinal root lesions, neuritis, automimmune diseases, general inflammation, chronic inflammatory conditions, arthritis, rheumatic diseases, lupus, osteoarthritis, general gastrointestinal disorders, colitis, gastric ulceration, duodenal ulcers, inflammatory bowel disorders, irritable bowel syndrome, pain associated with diarrhea, inflammatory eye disorders, inflammatory or unstable bladder disorders, psoriasis, skin complaints with inflammatory components, sunburn, carditis, dermatitis, myositis, neuritis, collagen vascular diseases, inflammatory pain and associated hyperalgesia and allodynia, neuropathic pain and associated hyperalgesia and allodynia, multiple sclerosis, demyelinating diseases, diabetes, diabetic neuropathy pain, causalgia, pain resulting from amputation or abscess, phantom limb pain, fracture pain, bone injury, direct trauma, HIV infection, acquired immune deficiency syndrome (“AIDS”), small pox infection, herpes infection, exposure to toxins or other foreign particles or molecules, invasive cancer, cancer, chemotherapy, radiotherapy, hormonal therapy, burns, congenital defect, dental pain, gout pain, fibromyalgias, encephalitis, chronic alcoholism, hypothyroidism, uremia and vitamin deficiencies, trigeminal neuralgia, stroke, thalamic pain syndrome, general headache, migraine, cluster headache, tension headache, mixed-vascular and non vascular syndromes, sympathetically maintained pain, deafferentation syndromes, asthma, epithelial tissue damage or dysfunction, disturbances of visceral motility at respiratory, genitourinary, gastrointestinal or vascular regions, wounds, burns, allergic skin reactions, pruritis, vasomotor or allergic rhinitis, or bronchial disorders, dysmenorrhoea, pain during labor and delivery, dyspepsia, gastroesophageal reflux, pancreatitis, and visceralgia.

Other disorders of sensory or sympathetic neuron dysfunction that can be treated with Huwentoxin-IV variants of the invention include asthma, cough, heart-burn, itch, dermatitis, bladder instability, and Reynaud's disease.

Huwentoxin-IV variants of the invention can be tested for their effect in reducing or alleviating pain using animal models, such as the SNL (spinal nerve ligation) rat model of neuropathic pain, carageenan induced hyperalgesia model, the Freund's complete adjuvant (CFA)-induced hyperalgesia model, the thermal injury model, the formalin model and the Bennett Model and other modes as described in U.S. Pat. Appl. No. 2011/0124711A1 and U.S. Pat. No. 7,998,980. Carageenan induced hyperalgesia and (CFA)-induced hyperalgesia are models of inflammatory pain. The Bennett model provides an animal model for chronic pain including post-operative pain, complex regional pain syndrome, and reflex sympathetic dystrophy.

Any of the foregoing animal models may be used to evaluate the efficacy of Huwentoxin-IV variants of the invention inhibitor in treating pain associated with the animal models. The efficacy can be compared to a no treatment or placebo control. Additionally or alternatively, efficacy can be evaluated in comparison to one or more known pain relieving medicaments.

Pharmaceutical Compositions

The Huwentoxin-IV variants of the invention can be formulated in a pharmaceutically acceptable vehicle or carrier. A suitable vehicle or carrier may be water for injection, physiological saline solution or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. These solutions are sterile and generally free of particulate matter, and may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable excipients as required to approximate physiological conditions, such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc. Suitable vehicles and their formulation and packaging are described, for example, in Remington: The Science and Practice of Pharmacy (21st ed., Troy, D. ed., Lippincott Williams & Wilkins, Baltimore, Md. (2005) Chapters 40 and 41).

Pharmaceutical compositions of the invention include formulations involving Huwentoxin-IV variants in sustained- or controlled-delivery formulations. These formulations may be achieved through use of for example injectable microspheres, bio-erodible particles, microemulsions, nanoparticles, nanocapsules, macroemulsions, polymeric compounds (such as polyesters, polyamino acids, hydrogels, poly(lactic acid), polyglycolic acid or ethylene vinylacetate copolymers), beads or liposomes, that may provide controlled or sustained release of the Huwentoxin-IV variants which can be delivered via depot injection, known to those skilled in the art. For example, hyaluronic acid or implantable drug delivery device may be used, having the effect of promoting sustained duration in the circulation.

Pharmaceutical compositions of the invention may be formulated for inhalation as a dry, inhalable powder. The inhalation solutions may also be formulated with a propellant for aerosol delivery, or a nebulizer.

Pharmaceutical compositions of the invention may be formulated for oral delivery. Huwentoxin IV variants that that are administered in this fashion may be formulated with or without carriers customarily used in the compounding of solid dosage forms such as tablets and capsules. A capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized. Additional agents can be included to facilitate absorption of the Huwentoxin-IV variants. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed. A pharmaceutical composition of the invention is preferably provided to comprise an effective quantity of one or a plurality of Huwentoxin-IV variants in a mixture with non toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or another appropriate vehicle, solutions may be prepared in unit-dose form. Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.

The Huwentoxin-IV variants of the invention may be prepared for use for parenteral (subcutaneous, intramuscular or intravenous), intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intra-ocular, intra-arterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices, or any other administration, particularly in the form of liquid solutions or suspensions; for buccal or sublingual administration such as in the form of tablets or capsules; or intranasally such as in form of powders, nasal drops or aerosols or certain agents; transdermally in a form of a gel, ointment, lotion, cream or dusting powder, suspension or patch delivery system with chemical enhancers to either modify the skin structure or to increase the drug concentration in the transdermal patch, or with agents that enable the application of formulations containing proteins and peptides onto the skin (WO98/53847), or applications of electric fields to create transient transport pathways such as electroporation, or to increase the mobility of charged drugs through the skin such as iontophoresis, or application of ultrasound such as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402). The composition also may be administered locally via implantation of a membrane, sponge or another appropriate material onto which the desired molecule has been absorbed or encapsulated.

In certain embodiments, where an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration.

The concentration of the Huwentoxin-IV variants of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15%, 20%, 30%, 40%, 50%, 60% or 70% by weight and will be selected primarily based on fluid volumes, viscosities and other factors, according to the particular mode of administration selected. The Huwentoxin-IV variants of the invention can be lyophilized for storage and reconstituted in a suitable vehicle prior to use. This technique has been shown to be effective with conventional protein preparations. Lyophilization and reconstitution techniques are well known in the art.

An exemplary pharmaceutical compositions of the present invention may comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, and may further include sorbitol, sucrose, Tween-20 and/or a suitable substitute thereof.

The appropriate therapeutically effective dose can be determined readily by those of skill in the art. Effective dose refers to an amount or dosage sufficient to produce a desired result, i.e. to partially or completely prevent, stop, inhibit, reduce, or delay the perception of pain associated with any painful medical condition. The effective amount may vary depending on the specific vehicle and Huwentoxin-IV variant selected, and is also dependent on a variety of factors and conditions related to the subject to be treated and the severity of the pain. For example, factors such as the age, weight and health of the subject to be administered with the pharmaceutical compositions of the invention as well as dose response curves and toxicity data obtained in preclinical animal work would be among those considered. A determined dose may, if necessary, be repeated at appropriate time intervals selected as appropriate by a physician or other person skilled in the relevant art (e.g. nurse, veterinarian, or veterinary technician) during the treatment period. The determination of an effective amount or a therapeutically effective amount for a given agent is well within the ability of those skilled in the art.

Thus, a pharmaceutical composition of the invention for intramuscular injection could be prepared to contain 1 ml sterile buffered water, and between about 1 ng to about 100 mg, about 50 ng to about 30 mg or about 5 mg to about 25 mg of a Huwentoxin-IV variant of the invention. Similarly, a pharmaceutical composition of the invention for intravenous infusion could be made up to contain about 250 ml of sterile Ringer's solution, and about 1 mg to about 30 mg or about 5 mg to about 25 mg of a Huwentoxin-IV variant of the invention. Actual methods for preparing parenterally administrable compositions are well known and are described in more detail in, for example, “Remington's Pharmaceutical Science”, 15th ed., Mack Publishing Company, Easton, Pa.

The present invention will now be described with reference to the following specific, non-limiting examples.

Example 1 Design and Generation of Huwentoxin-IV Variants

Single position amino acid scanning library substituting Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, and Tyr at every non-cysteine residue within the wild type Huwentoxin-IV (ECLEIFKACNPSNDQCCKSSKLVCSRKTRWCKYQI; SEQ ID NO: 1) derived from the venom of the Chinese bird spider, Ornithoctonus huwena was generated. The Huwentoxin-IV variants were encoded as HRV3C protease cleavable human serum albumin (HSA) fusion proteins in the following format from N— to C-terminus: His₆-HSA-(GGGGS)₄-HRV3C cleavage site-Huwentoxin-IV variant. Every variant peptide, following cleavage from HSA had a residual N-terminal GP from the cleavage site as well as a C-terminal GK which is the endogenous amidation recognition sequence. The single position variants were tested in fluorescence-based screening assays measuring their ability to inhibit Veratridine-induced membrane potential and hits were confirmed in Qpatch electrophysiology. The C-terminal GK residues in the recombinantly expressed cleaved Huwentoxin-IV variants were also substituted.

Combinatorial libraries were designed to test for additive effects of select single position hits in an attempt to generate Nav1.7 antagonists with further improved potency and selectivity profile compared to the native peptide. Two combinatorial libraries were produced, one that combined E1N, E4R, R26K, Y33W, Q34S, and G36I (library NV1D7L5), the other combined N13Q, S19Q, V23R, K27Y, R29K, and K37R (library NV1D7L6).

Construction of Expression Vectors

cDNAs encoding the designed Huwentoxin-IV variant polypeptides were generated using a gene assembly technology described in U.S. Pat. No. 6,521,427. Briefly, the amino acid sequences of the designed peptide variants were back-translated to DNA sequences using human-high frequency codons. The DNA sequence of each variant gene, together with a portion of vector DNA including the DNA cloning sites, was synthesized as multiple oligonucleotides, some of which contained degenerate codons, and assembled into full-length DNA fragments. The assembled DNA fragments were amplified by PCR and PCR products were subsequently cloned as a pool. Pooled PCR products were digested with the appropriate restriction enzymes and cloned into the designed expression vector in such as manner as to fuse each toxin variant gene to the signal peptide and the fusion partner contained in the vector. Standard molecular biology techniques were used to identify a positive clone for each designed variant. The plasmid DNA from these positive clones was purified and sequence confirmed before expressing each Huwentoxin-IV peptide variant.

Protein Expression

HEK293F cells maintained in 293 Freestyle™ media (Invitrogen) were transiently transfected with plasmids encoding Huwentoxin-IV variants using Freestyle™ transfection reagent (Invitrogen) according to standard protocols. Transfected cells were placed in a humidified incubator set at 37° C. and 8% CO₂ for 4 days shaking at 125 RPM. The supernatant was separated from the cells by centrifugation at 5,000 g for 10 minutes and filtered through a 0.2 μm filter and concentrated 10 and 50 fold using an Amicon Ultra Concentrator 10K (Cat #UFC901096), and centrifuging for approximately 10 minutes at 3,750 g.

Protein Purification

The secreted Huwentoxin-IV variant proteins were purified via IMAC using 1 ml H isTrap HP columns (GE Healthcare). The chromatography method was run using an AKTA Xpress and protein was eluted from the column using a step gradient of Imidazole. Peak fractions were pooled and digested overnight with HRV3C protease (EMD cat#71493; 1 unit/100 μg protein). Cleaved peptide was purified via RP-HPLC using a C18(2) column (Phenomenex, cat#00G-4252-N0). The chromatography method was run on a Dionex HPLC system and the bound peptide was eluted using a linear gradient of acetonitrile. Peak fractions were collected, pooled and lyophilized.

Lyophilized peptides were re-suspended in HEPES buffered saline, pH7.4 (10 mM HEPES, 137 mM NaCl, 5.4 mM KCl, 5 mM glucose, 2 mM CaCl₂, 1 mM MgCl₂). Absorbance was measured at 280 nm, and concentrations calculated using each peptide's extinction coefficient. Peptides were analyzed by non-reducing SDS-PAGE.

For scale-up, proteins were purified in IMAC using 5 ml HisTrap HP columns (GE Healthcare, cat#17-5248-02). The chromatography method was run using an AKTA Explorer or FPLC and protein was eluted from the column using a step gradient of Imidazole. Peak fractions were pooled and concentrated using Amicon Ultra-15 centrifugal concentrators (Millipore, cat# UFC901096) and dialyzed overnight against 2 changes of Dulbecco's phosphate buffered saline, pH7.2 (Invitrogen, cat#14190). The fusion was then digested overnight with HRV3C (EMD cat#71493; 1 unit/100 μg protein). The cleaved fusion was purified by IMAC using 5 ml H isTrap HP columns. The peptide was collected in the flow through fraction. Pooled peptide was concentrated and polished via RP-HPLC using a C18(2) column (Phenomenex, cat#00G-4252-N0). The chromatography method was run on an Agilent 1100 HPLC system and the bound peptide was eluted using a linear gradient of acetonitrile.

Each peak fraction was analyzed by RP-HPLC on an analytical C18(2) column (Phenomenex, cat#00G-4252-E0) using an acetonitrile linear gradient. Fractions with the same retention times were pooled and lyophilized. Lyophilized peptides were re-suspended in HEPES buffered saline, pH7.4 (10 mM HEPES, 137 mM NaCl, 5.4 mM KCl, 5 mM glucose, 2 mM CaCl₂, 1 mM MgCl₂). Absorbance was measured at 280 nm, and concentrations calculated using each peptide's extinction coefficient. Final peptides were analyzed by electrospray ionization mass spectrometry on a Waters system.

Example 2 Characterization of Huwentoxin-IV Variants Membrane Depolarization Assays

Ability of the generated Huwentoxin-IV variants to inhibit membrane depolarization induced by Nav1.7 agonist veratridine (3-Veratroylveracevine; Biomol, Catalog# NA125) was measured using FRET assay (fluorescence resonance energy transfer) on FLIPR® Tetra using DISBAC2(3) (Invitrogen, K1018) as an electron acceptor and PTS18 (Trisodium 8-octadecyloxypyrene-1,3,6-trisulfonate) (Sigma) as a donor by exciting the donor at 390-420 nm and measuring FRET at 515-575 nm.

HEK293F cells stably expressing the hNav1.7 channel under G418 selection (Invitrogen) were cultured in DMEM/F12 supplemented with glutamine, 10% FBS, 1% NEAAs, and 400 μg/ml G-418. 50 μl of harvested cells were plated at 25,000 cells/well into poly-lysine coated 384-well black clear bottom plates. The plates were incubated at room temperature (RT) for 15 min followed by an overnight incubation at 37° C. All incubations were done in the dark unless otherwise stated. The next day, the wells were washed 4 times with assay buffer, and resuspended in 25 μl of assay buffer (137 mM NaCl, 4 mM KCl, 2 mM MgCl₂, 2 mM CaCl₂, 5 mM Glucose, 10 mM HEPES). 2× stock (6 μM) of the PTS18 dye was prepared by suspending the dye in 10% pluronic F127 in DMSO at 1:1 (v/v ratio). 25 μl of the 2×PTS18 stock was added into the wells and the cells were stained for 30 min at RT, after which the dye was washed off with the assay buffer.

Huwentoxin-IV peptides were suspended at 3× their final concentration in the assay buffer containing 10 μM DISBAC2(3) and 400 μM VABSC-1 to suppress background fluorescence (Sigma, cat#201987). 25 μl/well of the suspended Huwentoxin-IV peptides were added onto each well, and incubated for 60 minutes at RT. Depolarization was induced by 25 μM final concentration of veratridine (by adding 25 μl/well of 75 mM (3×) stock solution), and the reduction in the mean intensity of FRET dye fluorescence was measured 30 seconds after adding the agonist. A 1.3× dilution of each measured Huwentoxin-IV peptide occurred after adding veratridine by convention, the concentration at the beginning of the FLIPR® Tetra assay is reported. Tetracaine, TTX, Protoxin-II and Huwentoxin-IV are established sodium channel blockers and were used as controls in each experimental series.

Fluorescence counts for each well were converted to % inhibition by normalizing the signal to the negative control (response to agonist veratridine alone) and positive control (response to veratridine in the presence of 10 μM tetracaine)

For measurements, “spatial uniformity correction” (all fluorescence traces are normalized to the average initial starting intensity) and “subtract bias value” (subtract the initial starting intensity from each trace) are turned on in FLIPR® Tetra.

For screening mode, no averaging was performed and each uploaded data point represents the response in an individual well.

For concentration-response mode, all individual data points were used in a non-linear least-squares procedure to find the best fit to a Hill function using Origin software (Microcal). IC₅₀ values were extrapolated from the resultant fitted curve.

The mean and standard deviations of the positive (P±dP) and negative (N±dN) controls were used to calculate the amount of block (B) in a well with a response (R) as follows:

$y = {100\% \left( \frac{N - R}{N - P} \right)}$

The screening window (a measure of the data quality) is defined as:

$z^{1} = {1 - {3{\frac{{\delta \; N} + {\delta \; P}}{N - P}}}}$

Assay plates were accepted if (1) the screening window based on the controls was z′>0.5, and (2) the potency of control antagonists for that day were within ±0.5 log units of their historical mean.

Selectivity of Huwentoxin-IV variants were assessed by ability of the variants to inhibit NaV1.2-induced membrane depolarization using HEK293F cells stably expressing the hNav1.2 channel under G418 selection (Invitrogen cat #11330) as described for Nav1.7, except that depolarization was induced by about 8.35 μM final concentration of veratridine (by adding 25 μl/well of 25 μM (3×) stock solution). Selectivity was measured as a ratio of IC₅₀ (Nav1.2)/IC₅₀(Nav1.7).

QPatch Assay

HEK293 cells stably expressing human Nav1.7 were cultured in DMEM/F-12 media (1:1), supplemented with 10% fetal bovine serum, 400 μg/mL Geneticin and 100 μM NEAAs (all reagents from Invitrogen). The cells were maintained at 37° C. and in 5% CO₂ and assayed upon reaching ˜70-90% confluency. Before testing in QPatch (Sophion), cells were first dissociated using 0.05% trypsin (5 min at 37° C.), resuspended in CHO—S—SFM media (Life Technologies) and gently triturated to break up cell clumps. Cell density was adjusted to 1−2×10⁶/mL with the same media and cells were transferred to a cell “hotel” in QPatch HT and used in experiments for several hours.

For giga-ohm seal formation and whole-cell patch clamp recording, the extracellular solution contained 137 mM NaCl, 5.4 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 5 mM glucose, and 10 mM HEPES, pH=7.4 and osmolarity=315 mOsm. The intracellular solution contained 135 mM CsF, 10 mM CsCl, 5 mM EGTA, 5 mM NaCl and 10 mM HEPES, pH=7.3 and osmolarity=290 mOsm.

The voltage protocol used in the assay was as follows. From a holding potential of −75 mV, cells were first hyperpolarized to −120 mV for 2 sec and then depolarized to 0 mV for 5 ms before returning to the holding potential (−75 mV). This protocol was repeated once every 60 sec during liquid applications (see below). Cells were otherwise held at −75 mV when the above voltage protocol was not executed.

Upon establishment of the whole-cell recording configuration, a total of five applications of the extracellular solution (all containing 0.1% bovine serum albumin (BSA) with or without test compound, except for the last application, which contained 1 μM TTX without BSA) were made on to cells being recorded. The first application contained only the control buffer (5 μl). The voltage protocol was executed 10 times (for a total duration of 10 min) five sec after the application. The next three applications (5 μl each) contained a test compound (same compound at the same concentration for all three applications) or control buffer (for control cells only). Five seconds after each of these applications, the voltage protocol was again executed 10 times (also once per min). The last application contained 1 μM TTX (composed of three 10 μl sub-applications, each separated by 2 sec), five seconds after which the same voltage protocol was executed twice to obtain the baseline current.

Currents were sampled at 25 kHz and filtered at 5 kHz with an 8-pole Bessle filter. The series resistance compensation level was set at 80%. For each cell, the peak current amplitude at 0 mV for each current trace in the first four liquid applications was first subtracted from that of the last trace in the presence of TTX and then normalized to that of the last trace in the first (control buffer) application as % inhibition. To control for current rundown, this (% inhibition) value for each cell in the presence of a test compound was further normalized to the average % inhibition value for control (typically 5-6) cells in the same experiment. The mean value of the last two such values in the last compound application (i.e., the corrected % inhibition value for each concentration of a test compound) was used in concentration response calculations. All experiments were performed at room temperature (˜22° C.). Data are expressed as mean±se.

For reference compounds, results obtained from QPatch using this protocol, e.g., potency/kinetics, were in good accord with that from manual patch clamp.

Results

Library matrix for single substitution variants and their IC₅₀ values for Nav1.7 obtained using the depolarization assay on FLIPR® Tetra is shown in FIG. 1. Library matrix for single substitution variants and the IC₅₀ for Nav1.2 obtained using the depolarization assay on FLIPR® Tetra is shown in FIG. 2. Selectivity measured as a ratio of the obtained IC₅₀ for Nav1.2 to the obtained IC₅₀ for Nav1.7 of the single substitution variants is shown in FIG. 3. FIGS. 4 and 5 show sequences of the variants ranked by potency on Nav1.7 (FIG. 4) or selectivity (FIG. 5).

Select variants were tested in whole cell patch clamp experiments. The recombinant Huwentoxin-IV and Huwentoxin-IV variants were tested against Nav1.7 and Nav1.2 stably expressed in HEK293 cells using the QPatch assay described above. The IC₅₀ values obtained for each huwentoxin-IV variant for Nav1.7 and Nav1.2 using the whole cell patch clamp methods is shown in FIG. 6. Selectivity of the Huwentoxin-IV variants was calculated as above using the IC₅₀ values obtained from the whole cell patch-clamp experiments.

Using Huwentoxin-IV as a starting point single-position amino acid scanning library was designed to identify variants with improved potency or selectivity. Select single position variants with interesting properties were included into combinatorial libraries. Single-position variants that were used in the design of the combinatorial libraries included E1N, E4R, R26K, Y33W, Q34S, G36I, N13Q, S19Q, V23R, K27Y, R29K, and K37R (residue numbering according to SEQ ID NO: 267), all of which showed improvements in potency, selectivity or both. Additional single-position variants with improved properties include R26W (SEQ ID NO: 72), K27W (SEQ ID NO: 57), Q34F (SEQ ID NO: 6) and R29W (SEQ ID NO: 55). In addition, variants (E1N,E4R,R26K,Q34S) (SEQ ID NO: 5), (E1N,E4R,R26K,Q34S,G36I) (SEQ ID NO: 16), (E4R,R26K,Y33W,G36I) (SEQ ID NO: 48), (E1N,Y33W,Q34S,G36I) (SEQ ID NO: 83), (N13Q,R29K,K37R) (SEQ ID NO: 137), (E1N,R26K,Q34S,G36I) (SEQ ID NO: 192) and (R26K,Y33W) (SEQ ID NO: 46) identified from combinatorial libraries demonstrate improved potency and/or selectivity.

Example 3 Analgesic Activity of Huwentoxin-IV Following Intraplantar Administration in Rats Methods

Male Sprague-Dawley (CD) rats (Charles River, San Diego) weighing >300 grams were used in this study. Naive animals were trained for two days prior to the day of testing (in order to reduce the variability in responses). Training consisted of performing actual tests multiple times on each animal over a duration of ˜1 hour for each rat. Animals first received a mark with a Sharpie in the center of the dorsal aspect of the left paw just proximal to the toes to enable consistently testing the same site of the paw. Rats were then loosely wrapped in a towel leaving the hind paws uncovered, the left hind paw was placed in the Randall Selitto device with the maximum threshold set at 500 grams (Ugo-Basile Randall-Selitto Device, Analgesy-Meter) with the Sharpie mark just beneath the point of the cone on the test device that comes in contact with the paw and pressure was increased at a steady rate via electronic ramp with foot control until the animal responded. A ‘response’ for training followed the same criteria as that on the day of testing and consisted of any one of the following: 1) removal of the hind paw from the device, 2) a clear attempt at removal or 3) vocalization. Rats were tested up to 3 times consecutively unless they responded to a threshold greater than or equal to 100 grams. Over the course of the hour/day of training, 1-3 consecutive tests were made for each rat with 5-20 minutes apart.

For compound testing, trained, un-injured rats were tested once for the pre-compound thresholds. Animals were assigned into peptide- or vehicle-treated groups in such a manner as to produce comparable pre-administration threshold means. Experiments were conducted blind to treatment groups where possible. One test for each time point following injection (5, 10, 20, 30 45, 60 min) was taken and recorded.

Material Preparation and Local Administration in Hindpaw

Amidated Huwentoxin IV (Peptides International, Louisville, Ky.) was received in lyophilized form and reconstituted with HEPES-buffered saline, aliquoted and frozen at −20° C. Just prior to administration in the left dorsal hind paw, aliquots were thawed and diluted to appropriate concentrations using HEPES-buffered saline as the diluent. Because stress related to handling and paw injections may itself produce an increase in paw pressure threshold (stress-induced analgesia), rats were briefly anesthetized with isoflurane for the injection (5% induction; 2-3% maintenance). Animals were injected s.c. (100 μL of peptide solution or vehicle) in the dorsal aspect of the paw with the needle inserted left of center toward the ankle such that the tip of the needle ended just underneath the Sharpie mark in the center of the dorsal paw proximal to the toes.

Data Analysis

Gram thresholds were recorded and entered into Prism 5.01 (Graphpad Software Inc., LaJolla, Calif.) for graphing, generating area under the curve (AUC) values and statistical analysis. For comparison of gram values over time, a two-way ANOVA was used with a significance level of p<0.05. For generation of mean AUC values, the AUC for each rat in the peptide group was individually obtained and the mean AUC of the vehicle group was subtracted from it. The vehicle subtracted AUCs for each peptide-treated animal were averaged and compared either by Student's T-test or one-way ANOVA, each with a significance level of p<0.05.

Results

Huwentoxin-IV administered locally into the dorsal aspect of the paw produced a dose-dependent increase in paw pressure threshold in the Randall-Selitto test. Three and 30 nmoles Huwentoxin IV, but not 0.3 nmoles, increased thresholds significantly above those observed for vehicle-treated animals (FIG. 7). AUCs were significantly different between all 3 peptide treated groups (with mean vehicle AUC subtracted from the AUC for each animal) (FIG. 8). Some local edema was also noted following administration of each dose of Huwentoxin-IV. Similar edema was not noted in the vehicle injected rats.

Example 4 Molecular Modeling of Huwentoxin-IV Interaction with Nav1.7 NMR Structure Determination

All NMR experiments were performed using Bruker Avance 600, 700, or 950 MHz spectrometers. The peptides were dissolved in aqueous buffer containing 10% D₂O. The buffer maintained a pH of 6.7 using 20 mM phosphate, 0.1 mM dEDTA, and 0.002% NaN₃. All spectra were collected at 298 K, unless otherwise stated. Individual residue spin systems were assigned using TOCSY (Bax and Davis, Mag. Reson. 1985, 65, 355-360) spectra using spin-lock (MLEV) with mixing times of 75 ms. Sequential residue assignments were made from NOESY (Jeener et al., J. Chem. Phys. 1979, 71, 4546-4553; Kumar et al., Biochem. Biophys. Res. Commun. 1980, 95, 1-6) experiments collected with a mixing time of 150 ms. In addition, ¹⁵N-HSQC (Bodenhausen et al., Chem. Phys. Lett. 1980, 69, 185-189) experiments aided assignment, and Cysteine oxidation states were elucidated via ¹³C-HSQC spectra using routine methods (Cavanagh et al., Protein NMR Spectroscopy: Principles and Practice 1995 Academic Press). Shifted sinebell squared weighting and zero filling was applied before Fourier transformation using NMRPipe (Delaglio et al., J. Biomol. NMR 6, 277-293, 1995) during data processing. Interproton distance restraints were derived from through-space interactions observed in the NOESY spectra, and automatically assigned by CYANA (Guntert et al., J. Mol. Biol. 273, 283-298, 1997). In addition, peptides containing W32 that showed significant (>0.2 ppm) ring current anisotropy on neighboring amino acids have aromatic side-chain restraints applied. The applications PREDITOR (Berjanskii et al., Nuc. Acid. Res. 2006, 34, W63-W69) and DANGLE (Cheung et al., J. Mag. Reson. 202, 223-233, 2010) were used to predict phi and psi angle ranges based on chemical shift data. Backbone omega angle restraints were set to 180°. Based on data derived from the NOESY and ¹³C—HSQC experiments disulfide bonds were fixed between, C9-C24, C2-C17, and C16-C31.

Homology models of the peptides were used as input (Cycle 1) to CYANA followed by six cycles of combined automated NOESY assignment and structure calculation. During each cycle 1000 conformers were calculated using a standard simulated annealing schedule with 10000 torsion angle dynamics steps per conformer followed by 50000 steps of energy minimization. Ensembles of 20 conformers with the lowest target function values were then used as input into an explicit water, distance restrained minimization refinement routine using MOE (Chemical Computing Group Inc., www://_chemcomp_com).

Molecular Dynamics

An NMR structure of native HwTx-IV (structure at Protein Data Bank http://_www_rcsb_org/pdb/home/home_do; pdb 1 MB6) was used as the starting point to characterize the stability of HwTx-IV using molecular dynamics simulations. In addition to simulations of the native HwTx-IV, simulations were performed to discern the importance of each of the three disulfide bonds and to determine the changes in peptide stability due to single alanine point mutations. To characterize the importance of the three disulfide bonds, separate molecular dynamics simulations (total of 7 simulations) were performed with the C2-C17, C9-C24, C16-C31, C2-C17/C9-C24, C2-C17/C16-C31, C9-C24/C16-C31 and C2-C17/C9-C24/C16-C31 cysteines converted into individual cysteine residues. To determine the effects of a single alanine point mutation an in silico molecular dynamics alanine scan (of all non-cysteine positions) was performed (total of 28 simulations).

For each molecular dynamics simulation, the HwTx-IV was solvated in explicit water (with a minimum of 12 Å padding) and neutralized to 0.1M NaCl. The protein was minimized and equilibrated for 50 ns using NAMD 2.8 [James et al., Journal of Computational Chemistry, 26:1781-1802, 2005). CHARMM 22 CMAP [MacKerell, Jr. et al., J Comput Chem 25: 1400-1415, 2004) parameters were used for the simulations with a multiple time stepping algorithm for evaluating electrostatics with bonded interactions computed every 1 fs, short-range non-bonded interactions computed every 2 fs, and long-range interactions computed every 4 fs. Long range electrostatic forces were evaluated using the particle mesh Ewald summation method with a grid spacing of less than 1 Å. Temperature was maintained at 300K using Langevin dynamics and a constant pressure of 1 atm was maintained using a Nose-Hoover Langevin piston. Periodic boundary conditions were assumed and non-bonded interactions were calculated using scaled 1-4 exclusion with shifting starting at 8 Å and a complete cut-off at 12 Å. Following simulation, the molecular dynamics trajectories were aligned based on the backbone C-alpha (CA) atoms and the root mean square deviation (RMSD) per residue calculated over the entire simulation relative to the initial NMR structure using Visual Molecular Dynamics (VMD) (Humphrey et al., J. Molec. Graphics, 1996, vol. 14, pp. 33-38).

Homology Modeling of Nav1.7 and Docking of HwTx-IV

A homology model of Nav1.7 Domain 2 (DII) segments S1-S4 was built with the structure NavAb (voltage-gated Na(+) channel from Arcobacter butzleri; structure at Protein Data Bank http://_www_rcsb_org/pdb/home/home_do; pdb 3RVY) as a template using the Modeller component in Discovery Studio 3.1 (Accelrys). The model was then further refined to generate a resting state Nav1.7 structure. S4 was manually moved down into a resting state configuration, the S1-S2 and S3-S4 loops were regenerated and the entire model was energy minimized. Native HwTx-IV was manually docked into the Nav1.7 homology model based on the results of the alanine scan of HwTx-IV inhibition against Nav1.7 and on published Nav1.7 mutations that effect HwTx-IV binding (Xiao et al., J Biol. Chem. 286:27301-10, 2011. Epub 2011 Jun. 9.).

Following the manual docking, the entire Nav1.7 DII S1-S4 with docked HwTx-IV system was minimized and an implicit membrane molecular dynamics simulation performed using the CHARMm forcefield with Generalized Born Implicit Membrane (Discovery Studio (Spassov et al., J. Phys. Chem. B, 106, 8726-8738, 2002) to further refine the docked structure.

Results Molecular Dynamic Simulations

A series of molecular dynamics simulations were conducted to help understand the molecular basis for changes in the activity of the HwTx-IV mutants that lead to significant loss of activity (F6A, P11A, D14A, L22A, S25A, W30A, K32A, Y33A) or channel selectivity (K18A, R26A and K27A) based on structural changes of the toxins alone. The previously generated NMR structure for HwTx-IV (pdb code 1 MB6) was used as a template for building the various alanine mutant peptides and each toxin variant was subjected to 50 ns of molecular dynamics simulations.

The average CA RMSD of native HwTx-IV peptide was only 1.007 Å indicating a highly stable peptide. Molecular dynamics simulations revealed that only W30A (FIG. 9 g), F6A (FIG. 9 b) (which normally form a pi-pi interaction) and L22A (FIG. 9 e) could influence the core stability of HwTx-IV. All other loss of function mutants exerted little to no effect on core stability. On the contrary, all loss of function mutants, as well as the mutants that differentially affected Nav1.7 and Nav1.2 activity were able to influence the flexibility of the loop regions. For example, W30A (FIG. 9 g), F6A (FIG. 9 b) and L22A (FIG. 9 e), increased the flexibility of loops 3 and 4, K32A (FIG. 9 h) increased loop 3 flexibility and D14A (FIG. 9 d) and P11A (FIG. 9 c) showed a pronounced increase in loop 2 flexibility. K27A (FIG. 10 c) and R26A (FIG. 10 b) were found to increase loop 4 flexibility. K18A (FIG. 10 a) and S25A (FIG. 9 f) mutations did not impact the flexibility of any loops.

NMR

To gain additional insight into the structural features of HwTX-IV and to directly test some of the main predictions of the molecular dynamic simulations we determined the NMR structure of recombinant WT HwTX-IV and compared it to the structure of W30A and K32A.

Despite the complete loss of activity measured in the QPatch and binding assays, but largely in keeping with the molecular dynamic simulations, W30A and K32A exhibit a similar global structure to WT recombinant HwTX-IV. Although interproton NOESY's and backbone chemical shift values indicate W30A, K32A, and the wild type peptides have very similar folds and structure, local differences are apparent near the solvent exposed face of the twisted beta-sheet. These differences include the observation of strong ring current anisotropy within a 5 angstrom radius of W30 in the K32A and wild type peptides. This anisotropy, most notably affecting F6 and T28, is indicative of a close spatial interaction that may affect the conformation/dynamics of the β-turn as well as the orientations of the side-chains. The solution structures imply another local difference, based on side-chain geometry, a potential cation-π interaction between the protonated amine of K32 and the π electrons of Y33, available to the W30A and wild type peptides. The side-chains of the five residues involved with these local differences, F6, T28, W30, K32, Y33 all reside in close proximity to each other lending to the aforementioned intra-molecular interactions as well as to form a potential ‘pharmacophore’ for inter-molecular interactions with Nav1.7.

Homology Modeling and Docking

In order to explore the specific interactions made between HwTx-IV and the Nav1.7 channel, a homology model of Nav1.7 domain II (DII) voltage sensor domain (VSD; segments S1-S4) was constructed using NavAB as a template. The model was further refined to produce a resting state structure into which to manually dock the HwTx-IV peptide using available SAR data along with published channel mutation data (Xiao et al., Biol. Chem. 286:27301-10, 2011. Epub 2011 Jun. 9.).

The published channel mutation data suggested that HwTx-IV binds in the DII voltage sensor domain with interactions with the S1-S2 and S3-S4 loops (specifically with residues E753, E811, D816, and E818). The resulting docked structure is presented in FIG. 12, with the hydrophobic patch comprised of W30 and F6 along with the basic K32 residue oriented in the groove formed by Nav1.7 S1-S2 and S3-S4 loops. The docked model places the W30 and F6 hydrophobic patch interacting with the channel groove with corresponding hydrophobic residue M750. While charged interactions along the edge of the S1-S2 loop and S3-S4 loop allow the HwTx-IV to orient itself in the binding site. Specifically on S1-S2 loop, charge-charge interactions are made between K7-E753 and E4-K762 of the HwTx-IV and the Nav1.7 channel respectively. Likewise, a series of charge-charge interactions between the HwTx-IV and the S3-S4 Nav1.7 loop also occur, R26-D816, K27-818, and K32-E811.

Example 5 Design and Generation of Additional Huwentoxin-IV Variants

Two grafting libraries were generated based on the obtained Huwentoxin-IV variants NV1G387 (E1N, R26K, Q34S, G36I; NV1D2168, SEQ ID NO: 192) and NV1G327 (E1N, E4R, Y33W, Q34S; NV1D2163, SEQ ID NO: 3).

Peptides were recombinantly expressed as described in Example 2, and IC50 values were measured using FLIPR® Tetra and QPatch as described in Example 2. Selectivity to voltage-gated sodium channels Nav1.1, Nav1.2, Hav1.3, Nav1.4, Nav1.5 and Nav1.7 were assessed using both methods.

The variant NV1G387 (NV1D2168) demonstrated high selectivity towards Nav1.7 (FIG. 5) and was grafted with substitutions that in the original Huwentoxin-IV scan enhanced potency (Nav1.7 IC50>0.05 μM). The library design for NV1G387 is shown in Table 1.

TABLE 1 Favorable potency mutations E1N, R26K, Q34S, G36I NV1G387 (NV1D2168) ≦0.05 E1 C2 L3 E4 R, H, N, Q I5 Y F6 K7 A8 R, H, N, Q C9 N10 P11 S12 N13 A D14 Q15 R, N C16 C17 K18 Y S19 R, Q, P S20 R, D, N, P K21 R, H, F, N L22 R V23 C24 S25 I R26 K27 T28 R29 W30 C31 K32 Y33 W Q34 I35 G36 K37 R, F

The variant NV1G327 (NV1D2163) demonstrated high potency (FIG. 4) and was grafted with substitutions that in the original Huwentoxin-IV scan enhanced selectivity (in this experiment defined as >5× selectivity over NaV1.2 or undefined). The library design for NV1G327 is shown in Table 2.

TABLE 2 Favorable Selectivity Mutations E1N, E4R, Y33W, Q34S NV1G327 (NV1D2163) >5x or undef. >5x E1 C2 L3 E4 I5 F6 V, M M K7 Q A8 C9 N10 P11 R S12 N13 W, Q, S, G, I, P W, Q, G, I D14 Q, S, G, L, P Q, S, G, P Q15 D, E, W, V, P D, E, W, V, P C16 C17 K18 F, W, Q, P F, W, Q, P S19 Q Q S20 W, V W, V K21 W W L22 E, W, Q, A W, A V23 A A C24 S25 R26 K, H, D, W, T, G, A, V, I, P K, H, W, T, G, A, V, I, P K27 H, W, A, I, P H, W, A T28 K, L R29 H, D, W, N, G, L H, D, W, N, G W30 K, Y K, Y C31 K32 W, A Y33 Q34 I35 H G36 F, T, V, I I K37 R, Q, S, T, P R, S, P

FIG. 13A shows the sequences and FIG. 13B characteristics of mutants based on the NV1G387 (NV1D2168) scaffold. All values are IC₅₀ values in nM unless only a single point assay was performed. In the latter case, the percent inhibition (% I) achieved at a given peptide concentration is listed.

FIG. 14A shows sequences and FIG. 14B characteristics of mutants based on the NV1G327 (NV1D2163) scaffold. Values in FIG. 14B are as in FIG. 13B.

The Huwentoxin-IV variants from the bidirectional grafting libraries demonstrated improved selectivity and/or include variants

>NV1G559 (SEQ ID NO: 277) GPNCLEIFKACNPSNDQCCKSSFLVCSKKTRWCKYSIIK (E1N, R26K, Q34S, G36I, grafted with K21F) >NV1G566 (SEQ ID NO: 278) GPNCLEIFKACNPSNDQCCKSNKLVCSKKTRWCKYSIIK (E1N, R26K, Q34S, G36I, grafted with S20N) >NV1G611 (SEQ ID NO: 279) GPNCLRIFKACNPSNDQCCKSSKLVCSDKTRWCKWSIGK (E1N, E4R, Y33W, Q34S, grafted with R26D) >NV1G612 (SEQ ID NO: 280) GPNCLRIFKACNPSNDQCCKSSKLVCSRHTRWCKWSIGK (E1N, E4R, Y33W, Q34S, grafted with K27H) 

We claim: 1) An isolated Huwentoxin-IV variant comprising a sequence X₁CX₂X₃X₄FX₅X₆CX₇X₈X₉X₁₀X₁₁X₁₂CCX₁₃X₁₄X₁₅X₁₆X₁₇X₁₈CX₁₉X₂₀X₂₁X₂₂X₂₃X₂₄CKX₂₅X₂₆IX₂₇X₂₈ (SEQ ID NO: 265); wherein a) X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄, X₁₅, X₁₆, X₁₇, X₁₈, X₁₉, X₂₀, X₂₁, X₂₂, X₂₃, X₂₄, X₂₅, and X₂₆ are any amino acid; b) X₂₇ and X₂₈ are any amino acid or deleted; and c) the Huwentoxin-IV variant has an IC₅₀ value about 300×10⁻⁹ M or less for human Nav1.7 (SEQ ID NO: 263), with the proviso that the Huwentoxin-IV variant is not a polypeptide comprising a sequence shown in SEQ ID NO:
 1. 2) The isolated Huwentoxin-IV variant of claim 1, wherein a) X₄ is Y, V or I; b) X₈ is P or V; c) X₁₁ is D, P or W; d) X₁₉ is S or I; e) X₂₁ is Y, W, A, K or H; f) X₂₂ is T or V; g) X₂₄ is W or K; and h) X₂₅ is W, T, I or Y. 3) The isolated Huwentoxin-IV variant of claim 2, wherein a) X₁ is K, R, H, D, Y, F, N, Q, S, T, G, L, I, P or E; b) X₂ is R, F, W, N, S or L; c) X₃ is R, H, D, Y, N, Q, L, I, P or E; d) X₅ is R, W, Q, S or K; e) X₆ is R, E, Y, F, V or A; f) X₇ is K, R, E, Y, F, S, V or N; g) X₉ is R, F, Q, V or S; h) X₁₀ is H, D, Y, W, Q, S, T, G, A, V, L, I, P or N; i) X₁₂ is K, R, D, E, Y, W, N, T, A, L or Q; j) X₁₃ is R, Y, Q, S, T, G, L, I, P or K; k) X₁₄ is K, R, Y, F, N, Q, G, A, V, L, I, P or S; l) X₁₅ is R, H, D, Y, W, N, Q, T, V, I, P or S; m) X₁₆ is R, H, D, F, W, N, Q, S, T, G, A, L or K; n) X₁₇ is K, R, Y, F, W, P or L; o) X₁₈ is K, R, T, A, L or V; p) X₂₀ is K, W, G, A, I, R or D; q) X₂₃ is K, H, W, N, G, A, L or R; r) X₂₆ is K, R, Y, F, S, T, G, A, V, L, I or Q; s) X₂₇ is K, R, H, F, W, V, L, I, G or deleted; and t) X₂₈ is R, H, Y, F, W, N, G, V, P, K or deleted. 4) The isolated Huwentoxin-IV variant of claim 3, wherein the Huwentoxin-IV variant has an IC₅₀ of less than about 160×10⁻⁹ M for human Nav1.7. 5) The isolated Huwentoxin-IV variant of claim 4, wherein the Huwentoxin-IV variant comprises a polypeptide sequence of SEQ ID NOs: 3-222, 241-253 or 277-280. 6) An isolated Huwentoxin-IV variant comprising a sequence X₁CX₂X₃X₄FX₅X₆CX₇X₈X₉X₁₀X₁₁X₁₂CCX₁₃X₁₄X₁₅X₁₆X₁₇X₁₈CX₁₉X₂₀X₂₁X₂₂X₂₃X₂₄CKX₂₅X₂₆IX₂₇X₂₈ (SEQ ID NO: 265); wherein a) X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄, X₁₅, X₁₆, X₁₇, X₁₈, X₁₉, X₂₀, X₂₁, X₂₂, X₂₃, X₂₄, X₂₅, and X₂₆ are any amino acid; b) X₂₇ and X₂₈ are any amino acid or deleted; and c) the Huwentoxin-IV variant selectively inhibits Nav1.7, with the proviso that the Huwentoxin-IV variant is not a polypeptide comprising a sequence shown in SEQ ID NO:
 1. 7) The isolated Huwentoxin-IV variant of claim 6, wherein the Huwentoxin-IV variant comprises a polypeptide sequence of SEQ ID NOs: 5, 7, 12, 13, 16, 21, 25, 45, 46, 48, 55, 57, 58, 60, 61, 72, 74, 76, 78, 82, 83, 96, 109, 111, 113, 122, 127, 131, 134, 137, 141, 142, 149, 164, 165, 172, 175, 177, 178, 180, 182, 188, 189, 192, 198, 202, 204, 213, 215, 219 or 223-240. 8) An isolated Huwentoxin-IV variant comprising the polypeptide sequences shown in SEQ ID NOs: 3-253 or 277-355. 9) An isolated polynucleotide encoding the Huwentoxin-IV variant of SEQ ID NOs: 3-253 or 277-355. 10) A vector comprising the isolated polynucleotide of claim
 9. 11) A host cell comprising a vector of claim
 10. 12) An isolated Huwentoxin-IV variant comprising a sequence X₁CX₂X₃X₄FX₅X₆CX₇X₈X₉X₁₀X₁₁X₁₂CCX₁₃X₁₄X₁₅X₁₇X₁₈X₁₉CX₁₉X₂₀X₂₁TX₂₂ WCKYX₂₃X₂₄X₂₅X₂₆ (SEQ ID NO: 276); wherein a) X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄, X₁₅, X₁₆, X₁₇, X₁₈, X₁₉, X₂₀, X₂₁, X₂₂, X₂₃ and X₂₄ are any amino acid; b) X₂₅ and X₂₆ are any amino acid or deleted; and c) the Huwentoxin-IV variant has an IC₅₀ value about 300×10⁻⁹ M or less for human Nav1.7 (SEQ ID NO: 263), with the proviso that the Huwentoxin-IV variant is not a polypeptide comprising a sequence shown in SEQ ID NO:
 1. 13) The isolated Huwentoxin-IV variant of claim 12, wherein a) X₄ is Y, V or I; b) X₈ is P or V; c) X₁₁ is D, P or W; d) X₁₉ is S or I; e) X₂₁ is Y, W, A, K or H; and f) X₂₄ is I. 14) The isolated Huwentoxin-IV variant of claim 13, wherein a) X₁ is K, R, H, D, Y, F, N, Q, S, T, G, L, I, P or E; b) X₂ is R, F, W, N, S or L; c) X₃ is R, H, D, Y, N, Q, L, I, P or E; d) X₅ is R, W, Q, S or K; e) X₆ is R, E, Y, F, V or A; f) X₇ is K, R, E, Y, F, S, V or N; g) X₉ is R, F, Q, V or S; h) X₁₀ is H, D, Y, W, Q, S, T, G, A, V, L, I, P or N; i) X₁₂ is K, R, D, E, Y, W, N, T, A, L or Q; j) X₁₃ is R, Y, Q, S, T, G, L, I, P or K; k) X₁₄ is K, R, Y, F, N, Q, G, A, V, L, I, P or S; l) X₁₅ is R, H, D, Y, W, N, Q, T, V, I, P or S; m) X₁₆ is R, H, D, F, W, N, Q, S, T, G, A, L or K; n) X₁₇ is K, R, Y, F, W, P or L; o) X₁₈ is K, R, T, A, L or V; p) X₂₀ is K, W, G, A, I, R or D; q) X₂₂ is K, H, W, N, G, A, L or R; r) X₂₃ is K, R, Y, F, S, T, G, A, V, L, I or Q; s) X₂₅ is K, R, H, F, W, V, L, I, G or deleted; and t) X₂₆ is R, H, Y, F, W, N, G, V, P, K or deleted. 15) The isolated Huwentoxin-IV variant of claim 14, wherein the Huwentoxin-IV variant has an IC₅₀ of less than about 160×10⁻⁹ M for human Nav1.7. 16) A method of producing the isolated Huwentoxin-IV variant comprising culturing the host cell of claim 11 and recovering the Huwentoxin-IV variant by the host cell. 17) A pharmaceutical composition comprising the isolated Huwentoxin-IV variant of claim 8 and a pharmaceutically acceptable excipient. 18) A method of treating pain in a subject, comprising administering to the subject an effective amount of the Huwentoxin-IV variant of claim 8 to treat the pain. 